The proposed studies address the molecular mechanism of thyroid hormone action in the anterior pituitary. While much has been learned about how thyroid hormone stimulates gene expression, the molecular mechanism underlying thyroid hormone inhibition of gene expression is less well understood. In the thyroid gland, hormone synthesis is regulated by thyroid-stimulating hormone (TSH), which is secreted from the anterior pituitary gland. TSH expression in the pituitary, in turn, is controlled by thyroid hormones which feedback and inhibit the expression of TSH subunit genes, alpha and beta. Although most patients with thyroid disease have normal regulation of TSH secretion, patients with thyroid hormone resistance syndromes, due to mutations in the beta isoform of the thyroid hormone receptor (TR), have dysregulated TSH secretion which is central to the pathogenesis of the disorder. A through understanding of the mechanisms of thyroid hormone inhibition in normal and diseased states, therefore, is warranted. In this proposal, a comprehensive evaluation of pituitary thyroid hormone inhibition with four specific aims is planned. First, the physiologic significance of negative thyroid hormone response elements (nTREs) defined in vitro will be validated in vivo utilizing human TSH-beta luciferase reporter gene constructs in transgenic animals. Second, the role of TR isoforms on negative and positive thyroid hormone regulation will be explored. Human common glycoprotein alpha, TSH-beta, and TRH luciferase reporter gene constructs will be compared with positive TRE (pTRE) containing constructs for these studies. Isoform specificity for thyroid hormone inhibition and mapping of a TR-beta specific thyroid hormone inhibitory domain will be investigated using wild type and chimeric alpha and beta TRs. Third, the effect of RXR isoforms on the structure and function of TR complexes bound to nTRE of the TSH-beta subunit gene will be examined. Finally, the structure and function of TR- beta in patients with selective pituitary resistance to thyroid hormone action (PRTH) will be determined. Each aim is designed to answer a central question about thyroid hormone inhibition, and will, therefore, provide significant insight into the mechanism of thyroid hormone action in the pituitary. They will validate the location and function of nTREs in vivo ; determine whether specific TR isoforms mediate thyroid hormone inhibition and by what mechanism; examine the role, if any, RXRs play in thyroid hormone inhibition; and characterize mutations of TR-beta in patients with a clinically defined and selective pituitary resistance to thyroid hormone. When completed, these studies will provide unique insights into how thyroid hormone both stimulates and inhibits gene transcription and expand our understanding of thyroid hormone resistance syndromes in man.